Site-specific Labeling Research Articles

Rotameric heterogeneity of conserved tryptophan is responsible for reduced photochemical quantum yield in cyanobacteriochrome slr1393g3.

The red/green cyanobacteriochrome (CBCR) slr1393g3 exhibits a quantum yield of only 8% for its forward photoconversion significantly lower than other species from the same CBCR subfamily. The cause for this reduced photoconversion is not yet clear, although in the related NpR6012g4 dark-state structural heterogeneity of a paramount Trp residue has been proposed to cause the formation of nonproductive subpopulation. However, there is no such information on the equivalent residue in slr1393g3, W496. Here we use solid-state NMR to explore all possible sidechain rotamers of this Trp residue and their local interactions at the atomic level. The indole nitrogen (Nε1) is used as an NMR probe, achieved by site-specific 15N-indole labeling of a quadruply Trp-deleted variant and trehalose vitrification technique. The data reveal a set of seven indole rotamers of W496 with four distinct environments for the Nε1-H group. Only a minority population of 20% is found to retain the π-stacking and hydrogen-bonding interactions with the chromophore in the dark state that has been assigned to account for complete forward photoconversion. Our results demonstrate the direct role of W496 in modulating the forward quantum yield of slr1393g3 via rearrangement of its sidechain rotameric conformations.

A Novel Cell- and Virus-Free SARS-CoV-2 Neutralizing Antibody ELISA Based on Site-Specific Labeling Technology.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the global spread of coronavirus disease 2019 (COVID-19), creating an urgent need for updated methods to evaluate immune responses to vaccines and therapeutic strategies. In this study, we introduce a novel cell-free, virus-free SARS-CoV-2 neutralizing antibody ELISA (NAb-ELISA), which is based on competitive inhibition of the receptor binding domain (RBD) of spike protein binding to the angiotensin-converting enzyme 2 (ACE2) receptor. In this method, site-specific biotinylated hACE2-Fc-Avi recombinant protein is immobilized onto a 96-well plate for capture, and the RBD-Fc-vHRP recombinant proteins serve as detection probes. Evaluation of sera from wild type (WT) or Delta RBD-immunized mice using the NAb-ELISA and pseudovirus neutralization tests (pVNTs) demonstrated strong correlations between assays (R2 = 0.91 and 0.90 for the WT and Delta groups, respectively). Additionally, the NAb-ELISA successfully detected cross-neutralizing activity in sera, though with slightly lower correlation to pVNT (R2 = 0.70-0.83). By employing NAb-ELISA instead of an indirect ELISA for hybridoma screening, five monoclonal antibodies (mAbs) with neutralizing activities against WT, Delta, and BA.2 pseudoviruses were obtained. This assay offers a straightforward, rapid, and safe approach to characterizing vaccine-induced antibody responses and mAb neutralization activity. Notably, the NAb-ELISA platform can be quickly adapted to assess neutralizing antibody responses against emerging mutant strains, addressing the rapid mutation of the virus.

A Dual-Purpose Non-Canonical Amino Acid for the Expanded Genetic Code: Combining Metal-Binding and Click Chemistry.

A rationally designed dual-purpose non-canonical amino acid (Trz) has been synthesised and successfully incorporated into a protein scaffold by genetic code expansion. Trz contains a 5-pyridyl-1,2,4-triazine system, which allows for inverse-electron-demand Diels-Alder (IEDDA) reactions to occur on the triazine ring and for metal ions to be chelated both before and after the click reaction. Trz was successfully incorporated into a protein scaffold and the IEDDA utility of Trz demonstrated through the site-specific labelling of the purified protein with a bicyclononyne. Additionally, Trz was shown to successfully coordinate a cyclometallated iridium(III) centre, providing access to a bioorthogonal luminogenic probe. The luminescent properties of the Ir(III)-bound protein blue-shift upon IEDDA click reaction with bicyclononyne, providing a unique method for monitoring the extent and location of the labelling reaction. In summary, Trz is a new dual-purpose non-canonical amino acid with great potential for myriad bioapplications where metal-based functionality is required, for example in imaging, catalysis, and photo-dynamic therapy, in conjunction with a bioorthogonal reactive handle to impart additional functionalities, such as dual-modality imaging or therapeutic payloads.

Two-Dimensional Infrared Spectroscopy Resolves the Vibrational Landscape in Donor–Bridge–Acceptor Complexes with Site-Specific Isotopic Labeling

Two-Dimensional Infrared Spectroscopy Resolves the Vibrational Landscape in Donor–Bridge–Acceptor Complexes with Site-Specific Isotopic Labeling

Antibody Modification via Lipoic Acid Ligase A-Mediated Site-Specific Labeling.

Enzymatic modification, particularly utilizing lipoic acid ligase (LplA), has emerged as a transformative approach in biopharmaceuticals, enabling precise and site-specific protein modifications. This review delves into the innovative applications of LplA in antibody modifications, including the creation of antibody-drug conjugates (ADCs) and the advancement of tag-free conjugation techniques. LplA's ability to facilitate the incorporation of bioorthogonal groups and its adaptability to various substrates underscores its versatility. Key developments include the successful generation of dual-labeled antibodies and the application of LplA in modifying antibody fragments. Additionally, the review explores the potential for LplA to enhance the therapeutic efficacy of ADCs through improved drug-to-antibody ratios and site-specific payload attachment. The implications of these advancements are significant, suggesting that LplA-mediated modifications could lead to more effective and targeted antibody-based therapies. This review aims to provide a comprehensive overview of LplA's role in expanding the possibilities of enzymatic conjugation, setting the stage for future research and clinical applications.

Enhancing substrate specificity of microbial transglutaminase for precise nanobody labeling

Streptomyces mobaraenesis transglutaminase (smTG) can be used for site-specific labeling of proteins with chemical groups. Here, we explored the use of modified smTG for the biosynthesis of nanobody-fluorophore conjugates (NFC). smTG catalyzes the conjugation of acyl donors containing glutamine with lysine-containing acceptors, which can lead to non-specific cross-linking. To achieve precise site-specific labeling, we employed molecular docking and virtual mutagenesis to redesign the enzyme's substrate specificity towards the peptide GGGGQR, a non-preferred acyl donor for smTG. Starting with a thermostable and highly active smTG variant (TGm2), we identified that single mutations G250H and Y278E significantly enhanced activity against GGGGQR, increasing it by 41 % and 1.13-fold, respectively. Notably, the Y278E mutation dramatically shifted the enzyme's substrate preference, with the activity ratio against GGGGQR versus the standard substrate CBZ-Gln-Gly rising from 0.05 to 0.93. In case studies, we used nanobodies 1C12 and 7D12 as labeling targets, catalyzing their conjugation with a synthetic fluorophore via smTG variants. Nanobodies fused with GGGGQR were successfully site-specifically labeled by TGm2-Y278E, in contrast to non-specific labeling observed with other variants. These results suggest that engineering smTG for site-specific labeling is a promising approach for the biosynthesis of antibody-drug conjugates.

Impact of site-specific conjugation strategies on the pharmacokinetics of antibody conjugated radiotherapeutics

Antibody radionuclide conjugates are an emerging modality for targeted imaging and potent therapy of disseminated disease. Coupling of radionuclides to monoclonal antibodies (mAbs) is typically achieved by applying non-site-specific labelling techniques. With the ambition of reducing variability, increasing labelling efficacy and stability, several site-specific conjugation strategies have been developed in recent years for toxin- and fluorophore-mAb conjugates. In this study, we studied two site-specific labelling strategies for the conjugation of the macrocyclic chelating agent, DOTA, to the anti-Leucine Rich Repeat Containing 15 (LRRC15) mAb DUNP19. Specifically, one approach utilized a DOTA-bearing peptide (FcIII) with a strong affinity for the fragment crystallizable (Fc) domain of the human IgG1 of DUNP19 (DUNP19LF-FcIII-DOTASS), while the other leveraged a chemo-enzymatic technique to substitute the N-linked bi-antennary oligosaccharides in the human IgG1 Fc domain with DOTA (DUNP19LF-gly-DOTASS). To assess if these methods impact the antibody's binding properties and targeting efficacy, comparative in vitro and in vivo studies of the generated DUNP19-conjugates were performed. While the LRRC15 binding of both radioimmunoconjugates remained intact, the conjugation methods had different impacts on their abilities to interact with FcRn and FcγRs. In vitro assessments of DUNP19LF-FcIII-DOTASS and DUNP19LF-gly-DOTASS demonstrated markedly decreased affinity for FcRn and FcγRIIIa (CD16), respectively. DUNP19LF-FcIII-DOTASS demonstrated increased blood and tissue kinetics in vivo, confirming loss of FcRn binding. While the ablated FcγR interaction of DUNP19LF-gly-DOTASS had no immediate impact on in vivo biodistribution, reduced immunotherapeutic effect can be expected in future studies as a result of reduced NK-cells interaction. In conclusion, our findings underscore the necessity for meticulous consideration and evaluation of mAb labelling strategies, extending beyond mere conjugation efficiency and radiolabeling yields. Notably, site-specific labelling methods were found to significantly influence the immunological impact of Fc interactions. Therefore, it is of paramount importance to consider the intended diagnostic or therapeutic application of the construct and to adopt conjugation strategies that ensure the preservation of critical pharmacological properties and functionality of the antibody in use.

Controlled labelling of tracer antibodies for time-resolved fluorescence-based immunoassays

Tracer antibodies, which are labelled with fluorescent or other type of reporter molecules, are widely employed in diagnostic immunoassays. Time-resolved fluorescence immunoassay (TRFIA), recognized as one of the most sensitive immunoassay techniques, utilizes tracers labelled with lanthanide ion (Ln) chelates. The conventional approach for conjugating isothiocyanate (ITC) Ln-chelates to antibodies involves random chemical targeting of the primary amino group of Lys residues, requiring typically overnight exposure to an elevated pH of 9–9.3 and leading to heterogeneity. Moreover, efforts to enhance the sensitivity of the assays by introducing a higher number of Ln-chelates per tracer antibody are associated with an elevated risk of targeting critical amino acid residues in the binding site, compromising the binding properties of the antibody. Herein, we report a method to precisely label recombinant antibodies with a defined number of Ln-chelates in a well-controlled manner by employing the SpyTag/SpyCatcher protein ligation technology. We demonstrate the functionality of the method with a full-length recombinant antibody (IgG) as well as an antibody fragment by producing site-specifically labelled antibodies for TRFIA for cardiac troponin I (cTnI) detection with a significant improvement in assay sensitivity compared to that with conventionally labelled tracer antibodies. Overall, our data clearly illustrates the benefits of the site-specific labelling strategy for generating high-performing tracer antibodies for TRF immunoassays.

In Vitro Encapsulation of Functionally Active Abiotic Photosensitizers Inside a Bacterial Microcompartment Shell.

Bacterial microcompartments (BMCs) are self-assembling, selectively permeable protein shells that encapsulate enzymes to enhance catalytic efficiency of segments of metabolic pathways through means of confinement. The modular nature of BMC shells' structure and assembly enables programming of shell permeability and underscores their promise in biotechnology engineering efforts for applications in industry, medicine, and clean energy. Realizing this potential requires methods for encapsulation of abiotic molecules, which have been developed here for the first time. We report in vitro cargo loading of BMC shells with ruthenium photosensitizers (RuPS) by two approaches─one involving site-specific covalent labeling and the other driven by diffusion, requiring no specific interactions between cargo molecules and shell proteins. The highly stable shells retain encapsulated cargo over 1 week without egress and preserve RuPS photophysical activity. This study is an important foundation for further work that will converge biological BMC architecture with synthetic chemistry to facilitate biohybrid photocatalysis.

Hetero-Diels-Alder and CuAAC Click Reactions for Fluorine-18 Labeling of Peptides: Automation and Comparative Study of the Two Methods.

Radiolabeled peptides are valuable tools for diagnosis or therapies; they are often radiofluorinated using an indirect approach based on an F-18 prosthetic group. Herein, we are reporting our results on the F-18 radiolabeling of three peptides using two different methods based on click reactions. The first one used the well-known CuAAC reaction, and the second one is based on our recently reported hetero-Diels-Alder (HDA) using a dithioesters (thia-Diels-Alder) reaction. Both methods have been automated, and the 18F-peptides were obtained in similar yields and synthesis time (37-39% decay corrected yields by both methods in 120-140 min). However, to obtain similar yields, the CuAAC needs a large amount of copper along with many additives, while the HDA is a catalyst and metal-free reaction necessitating only an appropriate ratio of water/ethanol. The HDA can therefore be considered as a minimalist method offering easy access to fluorine-18 labeled peptides and making it a valuable additional tool for the indirect and site-specific labeling of peptides or biomolecules.

Encapsulation of Transketolase into In Vitro-Assembled Protein Nanocompartments Improves Thermal Stability.

Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous in vitro reassembly studies, we first investigated the effectiveness of in vitro reassembly and cargo-loading of two size classes of encapsulins Thermotoga maritima T = 1 and Myxococcus xanthus T = 3, using superfolder Green Fluorescent Protein. We show that the empty T. maritima capsid reassembles with higher yield than the M. xanthus capsid and that in vitro loading promotes the formation of the M. xanthus T = 3 capsid form over the T = 1 form, while overloading with cargo results in malformed T. maritima T = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the T. maritima encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that in vitro loading of transketolase into the T. maritima T = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of in vitro assembled encapsulins with applications in cargo stabilization.

Hybrid Small-Molecule/Protein Fluorescent Probes.

Hybrid small-molecule/protein fluorescent probes are powerful tools for visualizing protein localization and function in living cells. These hybrid probes are constructed by diverse site-specific chemical protein labeling approaches through chemical reactions to exogenous peptide/small protein tags, enzymatic post-translational modifications, bioorthogonal reactions for genetically incorporated unnatural amino acids, and ligand-directed chemical reactions. The hybrid small-molecule/protein fluorescent probes are employed for imaging protein trafficking, conformational changes, and bioanalytes surrounding proteins. In addition, fluorescent hybrid probes facilitate visualization of protein dynamics at the single-molecule level and the defined structure with super-resolution imaging. In this review, we discuss development and the bioimaging applications of fluorescent probes based on small-molecule/protein hybrids.

Abstract 2588: Bioengineering of site-specific antibody labeling from basic research to spatial omics platforms

Abstract Over the last few years, spatial multi-omics platforms have emerged as a powerful tool for characterizing cancer biology in multiple dimensions. Notably, these systems typically rely on multiplex immunolabeling. Conventionally, multiplexing is accomplished through carefully selecting primary antibodies from different animal species, and in combination with their corresponding polyclonal secondary antibodies. However, given the practical complication related to the species availability of primary antibodies, the multiplexing labeling is still under constraint due to the necessity for matched secondary species labels and the availability. For decades, non-specific labeling of primary antibodies through amine- or thiol-reactive chemistries was widely performed. Evolving research and commercial products have sought to overcome this antibody-based multiplexing limitations by developing enzymatic reaction-directed chemical reaction or antibody targeting proteins that could contain affinity to the Fc or Fab regions of antibodies. However, these methods mentioned above have substantive limitations and side-effects on affinity, conjugation number, buffer incompatibilities, storage incompatibilities, insufficient conjugation, and uncontrolled stoichiometric labeling. Herein, we introduce an efficient, sustainable, reproducible site-specific antibody covalent labeling strategy through engineering anti-IgG secondary nanobodies. These infer the stability, high-affinity of species specific and easy-to-produce IgG binders with the permanence of controlled activation for proximity-driven intermolecular covalent conjugation, with a promise for efficient multiplexity. This labeling technology addresses antibody labeling challenges through currently available approaches, and would substantially enhance multiplexity in a wide variety of research and clinical settings. Citation Format: Kai Tao, Matthew J. Rames, MingChong Dai, Gulsu Sener, Samuel Olson, Srivathsan Ranganathan, Sila Ozdemir, Adam Oken, Michael Brasino, Randy Armstrong, Yu-Jui Chiu, Ece Eksi, Bruce Branchaud, Sadik Esener. Bioengineering of site-specific antibody labeling from basic research to spatial omics platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2588.

Nonperturbative Fluorogenic Labeling of Immunophilins Enables the Wash-free Detection of Immunosuppressants.

Immunosuppressants are clinically approved drugs to treat the potential rejection of transplanted organs and require frequent monitoring due to their narrow therapeutic window. Immunophilins are small proteins that bind immunosuppressants with high affinity, yet there are no examples of fluorogenic immunophilins and their potential application as optical biosensors for immunosuppressive drugs in clinical biosamples. In the present work, we designed novel diazonium BODIPY salts for the site-specific labeling of tyrosine residues in peptides via solid-phase synthesis as well as for late-stage functionalization of whole recombinant proteins. After the optimization of a straightforward one-step labeling procedure for immunophilins PPIA and FKBP12, we demonstrated the application of a fluorogenic analogue of FKBP12 for the selective detection of the immunosuppressant drug tacrolimus, including experiments in urine samples from patients with functioning renal transplants. This chemical methodology opens new avenues to rationally design wash-free immunophilin-based biosensors for rapid therapeutic drug monitoring.

Exploring the Sensitivity of Antibody-Drug Conjugate Efficacy to the Selection of Payload, Antibody, and Cell line.

Antibody-drug conjugates (ADCs) make up a growing class of targeted therapeutics with important applications in cancer treatment. ADCs are highly modular in nature and thus can be engineered to target any cancer type, but their efficacy is strongly influenced by the specific choice of payload, antibody, and target cell. Considering the number of possible antibody-payload combinations, ADC development would benefit from an efficient method to narrow the number of ADC compositions to those with the highest and most universal potency prior to assessing pharmacokinetics and pharmacodynamics in animal models. To facilitate the identification of optimal ADC compositions, we describe the use of photoreactive antibody-binding domain-drug conjugates (known commercially as oYo-Link) to enable the site-specific labeling of off-the-shelf antibodies. This approach allows for the rapid generation of ADCs with a drug-to-antibody ratio of ∼2 with no subsequent purification required. As a demonstration of this approach, ADCs were generated with different combinations of tubulin-inhibitor drugs (DM1, DM4, VcMMAE, and VcMMAF) and anti-EGFR antibodies (cetuximab, panitumumab, anti-EGFR clone 425, and anti-EGFR clone 528) and were delivered to three EGFR-expressing cell lines (A431, A549, and MDA-MB-231). Real-time cytolysis assays indicated that the most effective antibody varied based on the choice of cell line: cetuximab was most potent against A431 cells, while 425 and 528 led to the greatest cytotoxicity against A549 and MDA-MB-231 cells. These results did not correlate with differences in measured anti-EGFR binding affinity as cetuximab had the highest affinity across all three cell lines, while 425 and 528 had the lowest affinities for all three cell lines. Panitumumab, which had the second-highest anti-EGFR affinity, exhibited the least effective cytolysis across A431, A549, and MDA-MB-231 cells. By demonstrating that ADC potency toward a given target is dependent on both the antibody and drug chosen, these findings can guide the selection of ADCs for further in vivo analysis.

Site-specific albumin tagging with NIR-II fluorogenic dye for high-performance and super-stable bioimaging.

Synthetic near-infrared-II (NIR-II) dyes are promising for deep tissue imaging, yet they are generally difficult to target a given biomolecule with high specificity. Furthermore, the interaction mechanism between albumin and cyanine molecules, which is usually regarded as uncertain "complexes" such as crosslinked nanoparticles, remains poorly understood. Methods: Here, we propose a new class of NIR-II fluorogenic dyes capable of site-specific albumin tagging for in situ albumin seeking/targeting or constructing high-performance cyanine@albumin probes. We further investigate the interaction mechanism between NIR-II fluorogenic dyes and albumin. Results: We identify CO-1080 as an optimal dye structure that produces a stable/bright NIR-II cyanine@albumin probe. CO-1080 exhibits maximum supramolecular binding affinity to albumin while catalyzing their covalent attachment. The probe shows exact binding sites located on Cys476 and Cys101, as identified by proteomic analysis and docking modeling. Conclusion: Our cyanine@albumin probe substantially improves the pharmacokinetics of its free dye counterpart, enabling high-performance NIR-II angiography and lymphography. Importantly, the site-specific labeling tags between NIR-II fluorogenic dyes and albumin occur under mild conditions, offering a specific and straightforward synthesis strategy for NIR-II fluorophores in the fields of targeting bioimaging and imaging-guided surgery.

Immobilization of azide-functionalized proteins to micro- and nanoparticles directly from cell lysate

Immobilization of proteins and enzymes on solid supports has been utilized in a variety of applications, from improved protein stability on supported catalysts in industrial processes to fabrication of biosensors, biochips, and microdevices. A critical requirement for these applications is facile yet stable covalent conjugation between the immobilized and fully active protein and the solid support to produce stable, highly bio-active conjugates. Here, we report functionalization of solid surfaces (gold nanoparticles and magnetic beads) with bio-active proteins using site-specific and biorthogonal labeling and azide-alkyne cycloaddition, a click chemistry. Specifically, we recombinantly express and selectively label calcium-dependent proteins, calmodulin and calcineurin, and cAMP-dependent protein kinase A (PKA) with N-terminal azide-tags for efficient conjugation to nanoparticles and magnetic beads. We successfully immobilized the proteins on to the solid supports directly from the cell lysate with click chemistry, forgoing the step of purification. This approach is optimized to yield low particle aggregation and high levels of protein activity post-conjugation. The entire process enables streamlined workflows for bioconjugation and highly active conjugated proteins.Graphical

Recombinant humanized Fab fragments targeting the CFC domain of human Cripto-1

In the era of immunotherapy, the targeting of disease-specific biomarkers goes hand in hand with the development of highly selective antibody-based reagents having optimal pharmacological/toxicological profiles. One interesting and debated biomaker for several types of cancers is the onco-fetal protein Cripto-1 that is selectively expressed in many solid tumours and has been actively investigated as potential theranostic target. Starting from previously described anti-CFC/Cripto-1 murine monoclonal antibodies, we have moved forward to prepare the humanized recombinant Fabs which have been engineered so as to bear an MTGase site useful for a one-step site-specific labelling. The purified and bioconjugated molecules have been extensively characterized and tested on Cripto-1-positive cancer cells through in vitro binding assays. These recombinant Fab fragments recognize the target antigen in its native form on intact cells suggesting that they can be further developed as reagents for detecting Cripto-1 in theranostic settings.

Extending the toolbox for RNA biology with SegModTeX: a polymerase-driven method for site-specific and segmental labeling of RNA

RNA performs a wide range of functions regulated by its structure, dynamics, and often post-transcriptional modifications. While NMR is the leading method for understanding RNA structure and dynamics, it is currently limited by the inability to reduce spectral crowding by efficient segmental labeling. Furthermore, because of the challenging nature of RNA chemistry, the tools being developed to introduce site-specific modifications are increasingly complex and laborious. Here we use a previously designed Tgo DNA polymerase mutant to present SegModTeX — a versatile, one-pot, copy-and-paste approach to address these challenges. By precise, stepwise construction of a diverse set of RNA molecules, we demonstrate the technique to be superior to RNA polymerase driven and ligation methods owing to its substantially high yield, fidelity, and selectivity. We also show the technique to be useful for incorporating some fluorescent- and a wide range of other probes, which significantly extends the toolbox of RNA biology in general.

Abstract A068: Site-specific modification of nanobodies for ImmunoPET of Liver Cancer utilizing Self-labeling nanobody-tag pair (SLANT) technology

Abstract Visualization of tumor cells is critical in diagnosing, monitoring, and treating patients with cancer. Liver cancer has an 18 percent 5-year survival rate and is the third leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC), the most common type of liver cancer, only takes up 18F-fluorodeoxyglucose (18F-FDG), the most commonly used clinical positron emission tomography (PET) tracer, 50% of the time. As such, developing an HCC-selective imaging agent could improve our ability for earlier diagnosis, post-treatment surveillance, and ability to increase patient longevity and quality of life. Single domain antibodies (sdAbs), or nanobodies (Nb), are attractive scaffolds for radiopharmaceutical development because of their high target affinity and specificity, ready availability from recombinant expression in bacteria, and small size for rapid clearance, better tumor penetration, and same-day imaging. However, conventional conjugation chemistry that exploits cysteines or lysines found on the native protein can abrogate the binding site of the smaller Nbs. We have previously used sortase A-based site-specific conjugation to address these challenges; however, improvements were still possible. Here, we developed a novel self-labeling Nb-tag pair (SLANT) that allows fast and specific covalent labeling of a Nb based on peptide epitope binding. We used SLANT to engineer a heterodimeric Nb construct consisting of a Nb (HN3) specific for Glypican-3, a heparin sulfate proteoglycan that is overexpressed in 75-90% of all HCCs and our self-labeling Nb. This design allows for site-specific labeling of the heterodimeric (GPC3-specific) construct in a manner that avoids modification of the tumor antigen-specific protomer. Labeling is achieved via exposure of the fusion protein to a crosslinking peptide functionalized with a chelator (DOTA). This methodology produces labeled protein at over 90 percent yields within minutes compared to less than 30 percent yields utilizing enzymatic methods. Biolayer interferometry and radioligand saturation studies were done to determine the binding affinity of the conjugate for GPC3 pre- and post-Indium-111 labeling. SPECT/CT and biodistribution studies were done at 1h, 3hrs, and 24hrs with HepG2 and HepG2-GPC3- xenografts to assess conjugate specificity and ability to accumulate in GPC3+ liver tumors. Our conjugate exhibited a nanomolar affinity for GPC3 in vitro (17-36 nM). SPECT/CT and biodistribution studies showed specificity for GPC3 in HepG2 liver cancer xenografts (~5.5% IA/g at 1-hour post-injection (p.i.)) and minimal uptake in HepG2 GPC3- tumors (~0.62% IA/g at 1-hour p.i.). Our results highlight the development of a new labeling paradigm applied to GPC3+ liver cancer. This approach can be used for same-day imaging and the rapid development of SPECT and PET imaging tracers for different cellular markers on cancer cells, addressing known tumor heterogeneity. Citation Format: Stanley Fayn, Chino C Cabalteja, Woonghee Lee, Divya Nambiar, Kwamena E Baidoo, Freddy E Escorcia, Ross W Cheloha. Site-specific modification of nanobodies for ImmunoPET of Liver Cancer utilizing Self-labeling nanobody-tag pair (SLANT) technology [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A068.